Structural and Biochemical Investigations on Fe-Only Hydrogenases

仅铁氢化酶的结构和生化研究

• 批准号: 0328341
• 负责人: John Peters
• 金额: $ 28.4万
• 依托单位: Montana State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2006-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0328341&HistoricalAwards=false
• 关键词: Structural; Biochemical; Investigations; Fe; Only

The main objective of this projectl is to investigate the structural and catalyticproperties of Fe-Only hydrogenases. Hydrogenases catalyze reversible hydrogenoxidation and function physiologically to either recycle the reduced electron carriers thataccumulate during a fermentative metabolism or couple the oxidation of hydrogen to thegeneration of reduced electron carriers that can be utilized in energy yielding processes.The metal-containing hydrogenases are classified according to their metal content andinclude those that contain both Ni and Fe (NiFe-hydrogenases) and those that contain justFe (Fe-Only hydrogenases). These enzymes are of paramount importance in themetabolism of numerous microorganisms. In addition, since hydrogen is being exploredas a future alternative fuel source, there is considerable interest in the details of thesehydrogen producing biological catalysts. The studies will focus primarily on testing hypotheses thatwere developed as a result of the detailed structural analysis of the Fe-Only hydrogenase(CpI) isolated from the anaerobic soil microorganism Clostridium pasteurianum. The specific objectives are (1) additional structural and biochemical studies on CpI probing a number of remaining key questions with respect to the H Cluster structure and the role of the accessory [FeS] clusters; (2) site-specificamino acid substitution experiments on CpI to examine the role of the polypeptideenvironment of the H Cluster in catalysis and stability; (3) structural characterization ofthe second Fe-Only hydrogenase (CpII) from C. pasteurianum to ascertain its primarysequence and structural relationship to other Fe-Only hydrogenases and to probe thecontributions of the environment of the H Cluster and the accessory FeS clusters indefining hydrogen oxidation versus proton reduction activities in the Fe-Onlyhydrogenases. The above objectives will be pursued by a multidisciplinary approach combiningbiochemical methods, spectroscopic methods and structure determination by X-raydiffraction methods to answer some of the key remaining questions with respect to thestructure and function of Fe-Only hydrogenases.

本项目的主要目的是研究只含铁的氢酶的结构和催化性质。氢酶催化可逆的氢氧化，其生理功能是回收发酵代谢过程中积累的还原电子载体，或将氢的氧化与可用于能量产生过程的还原电子载体的生成相结合。含金属的氢酶根据其金属含量被分类，包括同时含有镍和铁的氢酶(NiFe-氢酶)和只含有Fe的氢酶(仅含Fe的氢酶)。这些酶在许多微生物的新陈代谢中起着至关重要的作用。此外，由于氢正在被开发为未来的替代燃料来源，人们对这些产生氢的生物催化剂的细节非常感兴趣。这些研究将主要集中在对从厌氧土壤微生物巴氏梭菌中分离出的纯铁氢酶(CPI)进行详细结构分析后提出的假设。具体目标是(1)对CPI进行更多的结构和生化研究，探索与H簇结构和辅助[FeS]簇的作用相关的一些剩余关键问题；(2)对CPI进行定点氨基酸替代实验，以研究H簇的多肽环境在催化和稳定性中的作用；(3)巴氏杆菌第二个仅铁氢酶(CpII)的结构特征，以确定其主要序列和结构与其他仅铁氢酶的结构关系，并探索H簇和辅助FeS簇环境对决定氢氧化与质子还原活性的贡献。上述目标将通过结合生化方法、光谱方法和X射线衍射法确定结构的多学科方法来实现，以回答关于仅含铁的氢酶的结构和功能的一些关键剩余问题。